1. Field of the Invention
The present invention relates to an apparatus and a method for repeatedly performing a nuclear magnetic resonance measurement on a sample while changing measurement conditions.
2. Description of the Related Art
Molecules with functional properties in a living body, such as proteins, have larger molecular weight than compounds used for medicine and include properties of large molecules.
Large molecules representing proteins have a molecular function in a solution. The molecular function may be impaired or promoted by combining with specific small molecules.
Binding of and interaction of a specific small molecule with a specific large molecule have been detected by use of various methods. Especially, a measurement using nuclear magnetic resonance (hereinafter referred to as an NMR measurement) allows information on the structure of a large molecule or information on the structure of a small molecule to be directly observed. In addition, such an NMR measurement allows for evaluation of dissociation constants of and reaction rates of a large molecule and a small molecule based on changes in spectrum to be measured for a molecule concentration and allows for analysis of interaction of a large molecule with a small molecule based on the structures of the large molecule and small molecule. International Publication Number WO 01/23889 discloses a method for performing an NMR measurement on the interaction of a protein with a small molecule.
Among large molecules used for an NMR measurement, a protein is produced by the following methods: a method for extracting from a living organism present in nature; a method for extracting from a large-scale expression system using Escherichia coli, etc. containing genes relating to production of a protein; a method using a cell-free expression system having the ability to abundantly express proteins without using a living cell; or the like. A method for radiolabeling (hereinafter referred to as labeling) with isotopes of hydrogen, carbon, and nitrogen, which are main elements of a protein, is used in some cases. The labeling includes a method for labeling by combining three elements of hydrogen, carbon, and nitrogen (which are main elements of a protein), a method for labeling all elements, and a method for selective labeling to label only atoms belonging to a specific amino acid residue, and the like. Irrespective of the type of the methods, the cost for the labeling process is high.
A nuclear magnetic resonance spectroscopy apparatus (hereinafter referred to as an NMR apparatus) typically includes a magnet for generating a static magnetic field B0 and a nuclear magnetic resonance probe arranged in a bore of the magnet. The nuclear magnetic resonance probe includes one or more coils used to apply a radio frequency magnetic field B1 to a target sample and detect a reaction (response) of the sample to the magnetic field.
Conventional nuclear magnetic resonance probes include a probe for measuring a stationary sample and a flow through probe. For the probe for measuring a stationary sample, a sample is placed in a glass tube or ampoule (hereinafter referred to as a sample tube), and the sample tube is set at a predetermined position in an NMR apparatus so as to perform a measurement of the sample.
In a conventional probe for measuring a stationary sample, an NMR measurement is performed while small molecules are titrated by using a sample tube having an opening, which allows for detection of changes in NMR spectrum in response to an increase in molecule concentration. However, a sample solution includes large molecules, small molecules for evaluation of an effect as an agent, and another reagent. Thus, once the sample solution contains small molecules with a certain molecule concentration, it is difficult to perform an NMR measurement with the sample solution containing small molecules with a molecule concentration smaller than the certain molecule concentration. The concentration of small molecules is, in general, measured as a parameter while the amount of large molecules in a sample solution of a predetermined amount is maintained to be constant. If a buffer solution is injected to reduce the concentration of the small molecules, the amount of the sample solution is increased. Reducing the amount of the sample solution to a predetermined amount also reduces the amount of the large molecules, resulting in a change in the measurement condition.
Titration of small molecules in a sample tube used in the NMR measurement increases the entire volume of a sample solution, which causes a change in the concentration of large molecules present in the sample solution and a change in the solution level of the sample solution. In order to reduce the changes, it is necessary that the volume of a solution to be dropped be as small as possible compared with the volume of the sample solution.
To reduce the volume of the solution to be dropped, it is necessary that the concentration of small molecules be increased. The maximum concentration of small molecules in a solution to be dropped, however, is determined based on the solubility of the small molecules. In general, the solubility of a substance varies depending on the type of a solvent and the temperature of a solution. Therefore, in an NMR measurement with a change in the concentration of small molecules due to the drop, the type of the solvent and the temperature of the solution influence stability of the concentration of large molecules.
On the other hand, International Publication Number WO 03/007009 discloses a flow through probe including a sample inlet port, a sample outlet port, and an internal tube extending between the sample inlet port and the sample outlet port. The internal tube includes a cell for holding a sample. A sample is placed into the sample inlet port, flows through the internal tube, and enters the cell. After being measured, the sample flows through the internal tube and is taken out of the probe.
A conventional flow through probe is used in combination with a robot type sample transfer system. Flow through probes each combined with a different sample transfer system are available in the market, for example, from Gilson, Inc. For such a system, samples which are each adjusted for different measurement conditions must be prepared in a plurality of vessels. The samples are passed through an apparatus in which the samples can be taken out of the vessels and are transferred to a flow through probe which has been already set. After the NMR measurement is completed on a sample, the sample is taken out of the probe.
A combination of a conventional flow through probe with a sample transfer system requires that samples adjusted for a plurality of different concentration conditions be prepared. Thus, the number of types of solutions containing large molecules with a certain concentration is required for the number of the types of measurement conditions. This increases the cost required for the samples.
In the case of unknown large molecules or unknown small molecules, the entire measurement needs to be repeated to evaluate functional properties of the molecules until a desired range of measurement conditions and a desired degree of changes in measurement conditions are confirmed.